Effects of Temperature and CO2 Concentration on Induction of Carbonic Anhydrase and Changes in Efficiency of Photosynthesis in Chlorella vulgaris 11h

The increase in carbonic anhydrase (CA) activity and the decreasein apparent K_m(CO₂) for photosynthesis induced by reducing CO₂concentration during the growth of Chlorella vulgaris 11h cellswere followed under different temperatures. Both changes wereaccelerated by raising the temperature and reached an optimumat 32–37?C. When the CO₂ concentration was lowered from3 to 0.04%, the rate of photosynthetic O₂ evolution at limitingCO₂ concentrations increased and reached a stationary levelafter 3 h. Under such conditions, the concentration of CO₂ dissolvedin the algal suspension decreased logarithmically (t_1/2=10 min)and reached a concentration in equilibrium with 0.04% CO₂ inair after ca. 2 h. When high-CO₂ cells grown with 3% CO₂ in air were transferredto various lower CO₂ concentrations, CA activity and apparentK_m(CO₂) for photosynthesis changed depending on the CO₂ concentration.The CO₂ concentration which gives one-half the maximum valuefor K_m(CO₂) and one-half minimum value foi CA activities wasabout 0.5%. The inverse relationship observed for the changesin CA activity and the affinity for CO₂ in photosynthesis supportsthe theory that CA loweres the apparent K_m(CO₂) for photosynthesisin Chlorella vulgaris 11h. (Received August 27, 1984; Accepted February 8, 1985)     

Factors Controlling Induction of Carbonic Anhydrase and Efficiency of Photosynthesis in Chlorella vulgaris llh Cells

When Chlorella vulgaris llh cells which had been grown in airenriched with 2–4% CO₂ (high-CO₂ cells) were bubbled withair containing ca. 400 ppm CO₂, illumination at an intensityas low as the light compensation point (350 lux) was sufficientto increase the photosynthetic rate under limiting CO₂ concentrations.The same treatment induced carbonic anhydrase (CA) activity.The induction of CA activity and increase in photosyntheticrate at limiting CO₂ concentrations were observed in the presenceof 10 µM DCMU which completely inhibits photosynthesis.These results indicate that photosynthetic electron transportis not involved in CA induction in Chlorella vulgaris llh cells.The parallelism between the changes in CA activity and the rateof photosynthesis under limiting CO₂ concentrations agree withthe previous conclusion that the transport of CO₂ from outsideto the site of CO₂ fixation is facilitated by CA and hence lowersthe apparent K_m(CO₂) for photosynthesis. (Received December 24, 1982; Accepted May 10, 1983)     

Effect of Glucose on the Induction of the Carbonic Anhydrase and the Change in K1/2(CO2 of Photosynthesis in Chlorella vulgaris llh

Increases in carbonic anhydrase activity and decreases in K_1/2(CO)₂for photosynthesis in Chlorella vulgaris llh, which are inducedby adaptation of cells to low CO₂, were suppressed by the additionof glucose to the growth medium. The results show that phenomenainduced by decreases in CO₂ are controlled by glucose or itsmetabolites. (Received July 11, 1990; Accepted December 25, 1990)     

不同理化因子对雨生红球藻CG-11碳酸酐酶活性的影响

以雨生红球藻CG-11为实验藻株,探讨在不同CO2、HCO3-、Zn2＋浓度以及pH和氮磷比例条件下,藻细胞的碳酸酐酶活性对这些理化因子的响应。结果表明,通入空气实验组的碳酸酐酶活性最高,为（75.20±1.53）U·mg-1（Chla）,通入5%CO2条件下的碳酸酐酶活性为（9.96±1.43）U·mg-1（Chla）;高浓度HCO3-对碳酸酐酶活性亦具有明显抑制作用,培养液中可溶性无机碳的浓度与碳酸酐酶活性呈负相关;在实验设置的pH范围内,pH9.0时碳酸酐酶活性最高,为（62.32±3.25）U·mg-1（Chla）;适当的氮磷比与Zn2＋浓度显著提高了雨生红球藻CG-11的生长速率,碳酸酐酶的活性亦有明显提高。     

Carbonic Anhydrase of Chlamydomonas: Purification and Studies on its Induction Using Antiserum against Chlamydomonas Carbonic Anhydrase

Carbonic anhydrase (EC 4.2.1.1 [EC] ; CA) was purified by affinitychromatography from cells of the unicellular green alga Chlamydomonasreinhardtii which had been grown photoautotrophically in ordinaryair. Antiserum raised in rabbit against this purified CA crossreactedwith Chlamydomonas CA but not with spinach leaf CA nor bovineerythrocyte CA. When the CO₂ concentration provided to the algalcells was decreased from 4% to the ordinary air level (0.04%),CA activity and the content of CA protein determined by theimmunodiffusion test showed parallel increases. In contrast,when the CO₂ concentration was raised from air level to 4% CO₂CA activity and its content expressed on the basis of culturevolume remained rather constant. These results indicate thatsynthesis of the CA protein is induced when the CO₂ concentrationis lowered from 4 to 0.04% during algal growth. On the otherhand, the synthesis of CA stops when CO₂ concentration is raisedfrom air level to 4%. (Received June 30, 1984; Accepted October 8, 1984)     

Effects of Ammonia on Respiration, Adenylate Levels, Amino Acid Synthesis and CO2 Fixation in Cells of Chlorella vulgaris 11h in Darkness

The effects of NH₄Cl on respiration, adenylate and free aminoacid levels as well as dark CO₂ fixation were investigated usingnitrogen-starved Chlorella vulgaris 11h cells with or withoutaddition of methionine sulfoximine (MSX), an inhibitor of glutaminesynthetase. Upon addition of NH₄Cl (1 mM) to the cells not treatedwith MSX, respiration was stimulated and the level of ATP droppedrapidly, while the levels of ADP and AMP increased. NH₄Cl alsostimulated amino acid synthesis, especially of glutamine, andmarkedly enhanced dark CO₂ fixation. Addition of NH₄Cl to MSX-treatedcells stimulated respiration and lowered the level of ATP, butdid not enhance glutamine synthesis and only slightly stimulateddark CO₂ fixation. ⁴On leave from Institute of Medical Science, Advance R &D Co. Minami-Hashimoto, Sagamihara, Kanagawa-ken 220, Japan (Received January 28, 1984; Accepted April 19, 1984)     

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263)

We have measured the exchange of ¹⁸O between CO₂ and H₂O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of ¹⁸O from CO₂ in the fluid outside the cells provides a method to study CO₂ and HCO₃⁻ kinetics in suspensions of algae that contain carbonic anhydrase since ¹⁸O loss to H₂O is catalyzed inside the cells but not in the external fluid. Low-CO₂ cells of Chlorella vulgaris (grown with air) were added to a solution containing ¹⁸O enriched CO₂ and HCO₃⁻ with 2 to 15 millimolar total inorganic carbon. The observed depletion of ¹⁸O from CO₂ was biphasic and the resulting ¹⁸C content of CO₂ was much less than the ¹⁸O content of HCO₃⁻ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO₃⁻ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10⁻⁴ s⁻¹ due to our experimental errors. The Fick's law rate constant for entry of CO₂ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO₂ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO₂ entry into the cell or both. The CO₂ hydration activity inside the cells was 160 times the uncatalyzed CO₂ hydration rate.     

Effect of Temperature on Starch Degradation in Chlorella vulgaris 11h Cells

Analysis of products formed in Chlorella vulgaris 11 h cellsduring photosynthesis in air containing 3,000 ppm ¹⁴CO₂ at varioustemperatures revealed that the level of ¹⁴C-starch was maximumaround 20–24?C and decreased with further rise in temperatureuntil 40?C, while ¹⁴C-sucrose greatly increased at temperaturesabove about 28?C. Elevating the temperature from 20 to 38?Cduring photosynthetic ¹⁴CO₂ fixation resulted in a remarkabledecrease in ¹⁴C in starch and a concomitant increase in ¹⁴Cin sucrose. This conversion of starch to sucrose when shiftingthe temperature from 20 to 38?C proceeded even in the dark.Hydrolysis of sucrose by rß-fructosidase showed that,irrespective of the experimental conditions, the radioactivitiesin sucrose were equally distributed between glucose and fructose.The enhancement of starch degradation with temperature risewas more remarkable than that of the activity of ribulose bisphosphatecarboxylase from the same cells. When Chlorella cells whichhad been preloaded with ¹⁴C-starch after photosynthesis for30 min at 20?C were incubated in the dark for an additional30 min at 20?C, ¹⁴C-starch was degraded by only about 4%. However,the values after 30-min dark incubation at 28, 32, 36 and 40?Cwere increased by about 10, 19, 36 and 50%, respectively. Duringthe temperature-dependent conversion of starch to sucrose, nosignificant amount of radioactivity accumulated in free glucoseand maltose. (Received October 27, 1981; Accepted January 9, 1982)     

Form of inorganic carbon utilized for photosynthesis in Chlorella vulgaris 11 h cells

The rate of photosynthetic ¹⁴CO₂ fixation in Chlorella vulgaris11h cells in the presence of 0.55 mM NaH¹⁴CO₃ at pH 8.0 (20?C)was greatly enhanced by the addition of carbonic anhydrase (CA).However, when air containing 400 ppm ¹⁴CO₂ was bubbled throughthe algal suspension, the rate of ¹⁴CO₂ fixation immediatelyafter the start of the bubbling was suppressed by CA. Theseeffects of CA were observed in cells which had been grown inair containing 2% CO₂ (high-CO₂ cells) as well as those grownin ordinary air (containing 0.04% CO₂, low-CO₂ cells). We thereforeconcluded that, irrespective of the CO₂ concentration givento the algal cells during growth, the active species of inorganiccarbon absorbed by Chlorella cells is free CO₂ and they cannotutilize bicarbonate. The effects observed in the high-CO₂ cellswere much more pronounced than those in the high-CO₂ cells.This difference was accounted for by the difference in the affinityfor CO₂ in photosynthesis between the high- and low-CO₂ cells. (Received May 19, 1978; )     

Identification of Distinct Internal and External Isozymes of Carbonic Anhydrase in Chlorella saccharophila

External carbonic anhydrase (CA) was detected in whole cells of alkaline-grown Chlorella saccharophila but was suppressed by growth at acid pH or growth on elevated levels of CO2. Internal CA activity was measured potentiometrically as an increase in activity in cell extracts over that of intact cells. Cells grown under all conditions had equal levels of internal CA activity. Two isozymes were identified after electrophoretic separation of soluble proteins on cellulose acetate plates. The fast isozyme was found in cells grown under all conditions, whereas the slow isozyme was found only in cells grown at alkaline pH. Western blot analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis using antibodies produced against the periplasmic form of CA from Chlamydomonas reinhardtii revealed a single band at 39 kD, which did not change in intensity between growth conditions and was associated only with proteins eluted from the fast band. The slow isozyme was inactivated by incubation of cell extract at 30[deg]C and by incubation in 10 mM dithiothreitol, whereas the internal form was unaffected. These results indicate that external and internal forms of CA differ in structure and their activities respond differently to environmental conditions.     

CO2 Excretion Across Isolated Perfused Crab Gills: Facilitation by Carbonic Anhydrase

Crab gill carbonic anhydrase is shown to facilitate the excretionof carbondioxide across isolated perfused gills. A techniquefor perfusing crab gills and assessing the metabolic viabilityof perfused gills is also described in detail. The techniqueis used to follow the disappearance of ¹⁴C label as HCO₃^–and CO₂ from internal perfusate passing through the gill. Theexcretion of the label increases with the flow rate of the externalperfusate across the outside of the gills. The addition of carbonican hydrase to the internal perfusate results in a two- to fourfoldincrease in the excretion of label while Diamox (acetazolamide)treatment decreases the excretion of label by half. It is alsosuggested that carbonic anhydrase, present in muscle tissuesof crabs, minimizes the disequilibrium of the hemolymph CO₂system as metabolically produced CO₂ leaves the tissues andenters the hemolymph. Parallels are drawn between the presenceof carbonic anhydrase in the crab gill system and the presenceof this enzyme in the respiratory organs of both aquatic andterrestrial animals.     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Carbonic Anhydrase Activity and CO2-Transfer Resistance in Zn-Deficient Rice Leaves

It has been reported that carbonic anhydrase (CA) activity in plant leaves is decreased by Zn deficiency. We examined the effects of Zn deficiency on the activity of CA and on photosynthesis by leaves in rice plants (Oryza sativa L.). Zn deficiency increased the transfer resistance from the stomatal cavity to the site of CO₂ fixation 2.3-fold and, consequently, the value of the transfer resistance relative to the total resistance in the CO₂-assimilation process increased from 10% to 21%. This change led to a reduced CO₂ concentration at the site of CO₂ fixation, resulting in an increased gradient of CO₂ between the stomatal cavity and this site. The present findings support the hypothesis that CA functions to facilitate the supply of CO₂ from the stomatal cavity to the site of CO₂ fixation. We also showed that the level of mRNA for CA decreased to 13% of the control level during Zn deficiency. This decrease resembled the decrease in CA activity, suggesting the possible involvement of the CA mRNA level in the regulation of CA activity.     

Acclimation to High CO(2) in Bean : Carbonic Anhydrase and Ribulose Bisphosphate Carboxylase

Young bean plants (Phaseolus vulgaris L. cv Seafarer) grew faster in air enriched with CO₂ (1200 microliters per liter) than in ambient CO₂ (330 microliters per liter). However, by 7 days when increases in overall growth (dry weight, leaf area) were visible, there was a significant decline (about 25%) in the leaf mineral content (N, P, K, Ca, Mg) and a drop in the activity of two enzymes of carbon fixation, carbonic anhydrase and ribulose 1,5-bisphosphate (RuBP) carboxylase under high CO₂. Although the activity of neither enzyme was altered in young, expanding leaves during the acclimation period, in mature leaves the activity of carbonic anhydrase was reduced 95% compared with a decline of 50% in ambient CO₂. The drop in RuBP carboxylase was less extreme with 40% of the initial activity retained in the high CO₂ compared with 50% in the ambient atmosphere. While CO₂ enrichment might alter the flow of carbon into the glycolate pathway by modifying the activities of carbonic anhydrase or RuBP carboxylase, there is no early change in the ability of photosynthetic tissue to oxidize glycolate to CO₂.     

Changes in the Carbonic Anhydrase Activity and the Rate of Photosynthetic O2 Evolution during the Cell Cycle of Chlorella ellipsoidea C-27

Rhythmical changes in carbonic anhydrase activity(CA) and inphotosynthesis were observed during the cell cycle of Chlorellaellipsoidea C-27 synchronized at various concentrations of dissolvedCO₂ (dCO₂ with a regime of 16 h of light and 8 h of darkness.At a constant low concentration of dCO₂ (11 {diaeresis}M), intracellularCA activity showed obvious fluctuations with a peak at 8 h afterthe initiation of illumination, while extracellular CA activity,located on the cell surface, showed only minor fluctuationsalthough the activity was as high as the maximum activity ofintracellular CA. In contrast, obvious changes in the activitiesof intra- and extracellular CA activities were not observedat a high concentration of dCO₂ (520 {diaeresis}M). The ratioof photosynthetic activity at limiting versus saturating concentrationsof dCO₂, which is indicative of the affinity of cells for CO₂,showed clear rhythmical changes during the cell cycle and theratio was higher in low-CO₂ cells than in high-CO₂ cells. Thechanges in the ratio seemed to reflect the changes in CA activity. When the cells that had been synchronized under high CO₂ conditionswere transferred to low CO₂ conditions at any given stage inthe cell cycle, CA activity was induced in every case but thecapacity for induction of CA was greater in young cells thanin mature cells. This result suggests that the capacity of cellsto induce CA over the course of the cell cycle is closely relatedto endogenous aging of the cell. (Received August 29, 1988; Accepted December 28, 1988)     

Mechanism of Inorganic Carbon Uptake in Chlorella saccharophila: The Lack of Involvement of Carbonic Anhydrase

The acid-tolerant green alga Chlorella saccharophila maintainedphotosynthesis and accumulated intracellular pools of inorganiccarbon over a a range of external pH from 4.0 to 7.5. This accumulationwas unaffected by treatment of cells with 10 mol m^–3 acetazolamide(AZA). Cells grown at alkaline pH had extracellular carbonicanhydrase (CA), but CA activity was repressed when cells weregrown at pH 5.0. Acid-grown cells retained a high affinity forCO₂, both at acid and alkaline pH, and the ability to accumulateinorganic carbon. Rates of photosynthesis of acid-grown cellsand alkaline-grown AZA-treated cells at pH 8.0 were 2.5-foldhigher than the rate of CO₂ supply from the uncatalysed dehydrationof , indicating that the cells can take up as a source of substrate for photosynthesis. Isotopic disequilibrium experiments with acid-grown cells maintainingsteady-state photosynthesis at pH 7.5 demonstrate that ¹⁴C from¹⁴CO₂ was taken up more rapidly than from H¹⁴. This uptake takes place against a concentration gradient. Theseresults demonstrate that C. saccharophila cells have activetransport systems for the uptake of both CO₂ and and both operate without the mediation of CA. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, Chlorella saccharophila, inorganic carbon accumulation     

岩溶环境因子对细菌胞外碳酸酐酶表达及活性的影响

以从西南岩溶生态系统分离出的一株细菌（编号GLRT102Ca）为例,研究了温度、pH、金属离子和阴离子等主要岩溶环境因子对其胞外碳酸酐酶（CA）表达及活性的影响。结果照乐,任实验温度（10℃～50℃）和pH（5.5～9.0）范围内,实验菌株均能表达出不同程度的胞外CA活性,其中20℃～30℃以及中性偏碱性条件下的胞外酶活性较高。钙、镁、锌、钻等4种金属离子以及SO4^2-、H2PO4^-、NO3^-、NO2^-、Cl^-、Br^-、I^-和HCO3^-等8种阴离子往实验浓度范围内一般都能促进胞外酶活性的表达,但表达较高活性所需的离子浓度因不同离子而异。以上结果为进一步研究微生物碳酸酐酶的岩溶作用提供了一定的理论依据。     

Sulfite: Preferential sulfur source and modifier of CO2 fixation in Chlorella vulgaris

Sulfite was added at the time of inoculation to a standard and to a sulfate deficient medium of Chlorella vulgaris. It was not only used as a sulfur source, but besides this, at concentrations <1.0 mmol l^–1, the growth yield was enhanced up to 30% compared to sulfate saturated conditions. Higher sulfite concentrations increasingly inhibited cell growth. Growth rate determinations indicated that the enhancement, and the inhibition respectively, were confined to the very beginning of culture growth; the time period during which the sulfite was not yet oxidized (5–10 h). In contrast, an increased CO₂ fixation rate/unit of protein, occurring up to 5.0 mmol l^–1 sulfite and a shift towards the -carboxylation pathway, are persisting at least during the growth period of 4 days. The preferential uptake of sulfite, also indicated by a marked increase in methionine content of algal protein, presumably causes an increase in thylakoidal sulfolipids, and is such modifying the CO₂ fixation.Abbreviations PGA 3-phosphoglyceric acid - APS adenosine 5-phosphosulfate - PEP phosphoenolpyruvate